Dissertations / Theses on the topic 'Mixed culture fermentations'

Thesis (MSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: The use of non-Saccharomyces yeasts together with the yeast S. cerevisiae in multistarter wine fermentations has emerged as a useful tool to modulate wine aroma and/or to decrease the concentration of undesirable compounds. However, upon inoculation, these yeast species do not co-exist passively, but interact in various ways. While competition for nutrients and the excretion of killer toxins in an antagonistic relationship are obvious and well established types of interactions, some studies have suggested the existence of other forms of cellular or molecular interactions. One of these includes physical cell-cell contact and to our knowledge, only one previous study has confirmed its existence in wine yeasts. Yeast interactions are also influenced by other factors, such as ethanol concentration, however some studies have highlighted the role that dissolved oxygen plays on the survival of non-Saccharomyces yeasts and their ability to compete for space with S. cerevisiae and little research has focused on this. This study aimed to investigate the occurrence of a physical cell-cell and/or metabolic interaction between S. cerevisiae and L. thermotolerans in mixed culture fermentations of synthetic grape must. For this purpose, fermentations in a Double Compartment Bioreactor (DCB) which separates yeast population through the use of a membrane were compared to mixed fermentations in the absence of the membrane, using the same reactor. Furthermore, the impact of oxygen supply on yeast behaviour was also assessed. Following mixed culture fermentations in a DCB, it was observed that the presence of S. cerevisiae led to a significant decline in viability in L. thermotolerans. This decline was significantly less prominent in mixed cultures where the cells were in indirect contact. Together, the data provided evidence for both cell-cell and metabolic interactions whereby S. cerevisiae had a strong negative influence on the growth of L. thermotolerans. However, it was also observed that L. thermotolerans had some negative impact on the growth of S. cerevisiae, leading to a reduction in biomass (when in indirect contact) and a reduced maximum CFU/mL compared to pure cultures. The data also suggest that direct physical contact may increase the production of glycerol and propanol, but this needs further investigation. By decreasing the frequency at which oxygen pulses were provided, a reduction in biomass and increase in fermentation duration was observed for all fermentations. However, this effect was somewhat reduced in mixed cultures. Here, no impact on fermentation duration was observed and the decrease in biomass was less compared to pure cultures. The impact of these oxygen pulses was also greater on L. thermotolerans. In the latter yeast’s pure culture a slight increase in glycerol was observed when less oxygen was provided and in general there appeared to be no impact on acetic acid production. Furthermore, there was little or no impact on volatile production, however, more repeats might reveal different results and therefore more research is needed to confirm these results. To our knowledge, this is the first study of its kind to confirm a physical cell-cell interaction between the yeast pair S. cerevisiae and L. thermotolerans.
AFRIKAANSE OPSOMMING: Die gebruik van nie-Saccharomyces gis saam met die gis S. cerevisiae in multi-inokuleringskulture het die afgelope paar jaar as n goeie hulpmiddel na vore gekom om wyn aroma te moduleer en/of om die konsentrasie van ongewensde verbindings te verminder. Sodra inokulasie plaasgevind het, het hierdie gis die potensiaal om op verskeie maniere teenoor mekaar te reageer. Kompetisie vir nutriente en die afskeiding van toksiese verbindings in n antagonistiese verhouding is alreeds goed beskryf in die literatuur. Somige studies het, alhoewel, die bestaan van ander vorme van sellulêre of molekulêre interaksies voorgestel. Een van hierdie sluit in n fisiese sell-sell interaksie en so ver as wat ons kennis strek, het nog net een studie van tevore so ‘n interaksie bevestig tussen wyn giste. Gis interaksies word ook beïnvloed deur ander faktore, soos byvoorbeeld etanol konsentrasie. Terwyl sommige studies die rol wat opgelosde suurstof speel in die oorlewing van nie-Saccharomyces gis en hulle vermoë om te kompeteer vir spasie met S. cerevisiae alreeds beklemtoon, het min navorsing al hierop gefokus. Hierdie studie het gestreef om die voorkoms van n fisiese sell-sell en/of metaboliese interaksie tussen S. cerevisie en L. thermotolerans in gemengde kultuur fermentasies van sintetiese druiwe sap te ondersoek. Vir hierdie doeleinde was fermentasies uitgevoer met behulp van ‘n Dubbel Kompartement Bioreaktor (DKB) wat gis populasies skei deur middel van ‘n membraan en hierdie was vergelyk met gemengde kultuur fermentasies sonder die membraan in dieselfde reaktor sisteem. Verder was die impak van suurstof toevoer op gis gedrag ook geassesseer. Na afloop van gemengde kultuur fermentasies in ‘n DKB, was daar waargeneem dat die teenwoordigheid van S. cerevisiae gelei het tot ‘n betekenisvolle afname in lewensvatbaarheid in L. thermotolerans. Hierdie afname was aansienlik minder in gemengde kulture waar die gis in indirekte kontak was. Saam verskaf hierdie data bewyse vir n sell-sell asook metaboliese interaksie waardeur S. cerevisiae ‘n sterk, negatiewe invloed op die groei van L. thermotolerans gehad het. Daar was egter ook waargeneem dat L. thermotolerans tot ‘n mindere mate ‘n negatiewe impak op die groei van S. cerevisiae gehad het en dat dit gelei het tot ‘n verlaging in biomassa (toe die gis in indirekte kontak was) en ‘n verlaagde maksimum CFU/mL in vergelyking met suiwer kulture. Die data dui ook aan dat fisiese kontak kon gelei het tot ‘n verhoging in gliserol en propanol produksie, maar hierdie kort verdere ondersoek. Deur die frekwensie te verminder waardeur suurstof pulse aan die fermentasies verskaf was, was ‘n verlaging in biomassa produksie en ‘n verlenging in fermentasie tydperk waargeneem. Hierdie tendense was waargeneem in almal, behalwe die gemengde kultuur fermentasies. Die effek van suurstof puls verlaging was minder op hierdie fermentasies aangesien daar geen impak op fermentasie tydperk was nie en die verlaging in biomassa minder was. Die impak van hierdie suurstof pulse was ook groter op L. thermotolerans. ‘n Klein toename in gliserol produksie was waargeneem in laasgenoemde gis se suiwer kultuur toe minder suurstof beskikbaar was en oor die algemeen was asynsuur onveranderd. Verder was daar ‘n klein of geen impak op vlugtige verbindings nie, alhoewel, meer herhalings mag verskillende resultate lewer en daarom is meer navorsing nodig om hierde resultate te bevestig. So ver as wat ons kennis strek is hierdie die eerste studie van sy soort om ‘n fisiese sell-sell interaksie tussen die gispaar S. cerevisiae en L. thermotolerans te bevestig.

In the last decades increasing attention has been paid on syngas, produced through the thermal gasification of biomass, for energy recovery. Syngas components, mainly CO and H2, can be used as substrate for the conversion into important chemicals and fuels, in this case specifically methane, by a wide range of microorganisms. This study focused on the exploitation of an anaerobic mixed culture, collected from an anaerobic sludge of the Denmark Technical University (DTU) Bioengineering laboratory, in the fermentation of syngas for the production of CH4. A series of enrichment experiments with different initial partial pressure of CO and H2 were performed, in mesophilic and thermophilic conditions. Both yields and production rates resulted to be higher in thermophilic conditions; experiments with H2 tended to be faster than those without and the mmols of produced methane, over fed carbon (mmol/mmol), were higher than when only CO was present as substrate. A second experimental test has also been executed in thermophilic conditions (since they better performed in the previous experiment) to better evaluate which reactions are those which mainly took place during the syngas fermentation process. Experiments were performed both in the absence and in the occurrence of 2-bromoethanesulfonic acid as an effective inhibitor of the methanogens. Results showed that acetate mainly came from Acetogenesis via CO, being however negligible, while methane was produced either by hydrogenotrophic methanogens either via H2/CO. Production of bio hydrogen was also present, and it is usually further consumed to produce CH4. To conclude, findings of this projects may be considered as a further step towards the knowledge of biogas production through the use of microbial catalysts, giving important information both about the optimal operating conditions and about microbial metabolism.

The removal of multiple substrates in a defined mixed culture process was investigated in the treatment of brewery wastewater. The study was conducted using both batch and a semi-continuous reactor system called self-cycling fermentation. Batch experiments were conducted using a synthetic brewery wastewater containing glucose, ethanol and maltose. Activated sludge from a municipal wastewater treatment plant was acclimatized in the synthetic brewery wastewater. The microbes capable of degrading this wastewater were analyzed by a combination of microscopy, spread plating, and Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and identified as Acinetobacter sp., Enterobacter sp. and Candida sp. From the pure culture batch experiments, it was determined that Enterobacter could degrade glucose and maltose but no ethanol, while Acinetobacter and Candida could degrade all three carbon sources. In mixed culture batch experiments, Enterobacter was dominant in degrading the sugar concentrations to levels permissible for Acinetobacter to degrade ethanol. PCR-DGGE was found to be effective in identifying the dominant species but selective carbon source plating was required to determine viability and track the population dynamics. Kinetic experiments were carried out in a semi-continuous, self-cycling fermentation process using the defined mixed culture in media containing glucose and various initial concentrations of ethanol and maltose. The overall rate of substrate removal was attributable to both the suspended culture and the biofilm formed during the process. A rate expression was developed for this system for the range of substrate concentrations tested. The data indicated that substrate removal by the suspended culture was a function of only the biomass concentration. However, substrate removal by the biofilm was found to be limited to the surface cells and determined to be a function of substrate concentration only.

Thesis submitted in fulfilment of the requirements for the degree of Doctor Technologiae: Chemical Engineering, Faculty of Engineering at the Cape Peninsula University of Technology - Cape Town, South Africa
Polycyclic aromatic hydrocarbons (PAHs) are one of the most common and recalcitrant environmental contaminants – known for their potential toxicity, mutagenicity, and carcinogenicity to humans. Biosurfactant application can enhance the biodegradation of PAHs. The main object of this work was to explore the novelty of biosurfactant produced by the isolated strains of Bacillus sp and Pseudomonas aeruginosa grown exclusively on Beta vulgaris, and the modification of the zeolites nanoparticles by the biosurfactant, for enhanced biodegradation of PAHs in soil. Novel biosurfactant-producing strains were isolated from hydrocarbon-contaminated environments, while several agrowaste were screened as primary carbon sources for the expression of biosurfactants, which were quantified using various standardized methods......

This thesis aimed at investigating a process based on anaerobic mixed culture fermentation (AMCF) of organic waste to produce carboxylates, molecular hydrogen and ethanol. The novelty of this study was to estimate the potential production of these bulk chemicals from selected renewable feedstocks and to experimentally investigate their production from the AMCF of three distinct types of feedstock without physicochemical pre-treatment: grass, a lignocellulosic substrate; cheese whey, a lactose-rich residue; and fish processing wastewater, a complex effluent containing proteins and fats. The estimation showed that AMCF of a small fraction (less than 1.6%) of the selected feedstock would be required to suffice the current production of carboxylates from oil-based feedstock, whilst up to 51% of the feedstock would be needed to satisfy the current production of ethanol and molecular hydrogen. In the experiments with AMCF of grass, low organic loading rates and long solids retention times were required to increase the substrate conversion because of the hard-biodegradability found in the substrate without any physicochemical pre-treatment. For the AMCF of cheese whey, the production of carboxylates decreased the pH to low levels and consequently inhibited the fermentation. For the AMCF of fish processing wastewater, the feedstock showed high biodegradability with production of biogas under batch condition and stable production of carboxylates under semi-continuous condition. The release of ammonium during the degradation of proteins was considered one of the main factors to avoid the pH drop when carboxylates were produced throughout the fermentation. In conclusion, the study showed that the feedstock nature and the operating condition play a key role in how to drive the AMCF to the production of these chemicals and avoid methanogenesis. Whilst hydrolysis limits the fermentation for hardly biodegradable feedstock, volatile fatty acids formation and pH inhibit the AMCF of more easily biodegradable feedstock.

Thesis (MSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Grape must gives rise to various stress conditions for the yeast inoculated for alcoholic fermentation. These include hyperosmotic stress due to the high initial sugar concentration and redox imbalances due to the fast depletion of oxygen. Under these stress conditions, Saccharomyces cerevisiae tends to produce glycerol as an osmoprotectant and to regenerate reducing equivalents. However, the production of glycerol often leads to increased acetic acid production. According to literature, it seems that many non-Saccharomyces yeasts have a different metabolic response to the above-mentioned stress conditions, especially since it has been found that they produce low levels of acetic acid. Only recently non-Saccharomyces yeasts were researched to be used as starter cultures in wine fermentations. It is found that they can confer beneficial characteristics to the resulting wine. However, most of the non-Saccharomyces yeasts lead to stuck fermentations as confirmed by this study. Therefore, if the positive characteristics of these yeasts were to be exploited in wine making they need to be inoculated together with S. cerevisiae. When two yeasts are inoculated together, they affect each other and consequently the wine. In this context, the aim of this study was to investigate the metabolic response to hyperosmotic stress during wine fermentation of the following wine-related non-Saccharomyces yeasts: Lachancea thermotolerans, Torulaspora delbrueckii and Starmerella bacillaris. Fermentations were performed in a synthetic grape must medium with pure cultures of the mentioned strains as well as mixed cultures of each non-Saccharomyces yeast with S. cerevisiae. The fermentation behaviour was monitored and concentrations of various wine-related metabolites were determined. Concerning polyol concentrations, S. cerevisiae produced only glycerol while the non-Saccharomyces yeasts also produced other polyols. The low production of acetic acid in the non-Saccharomyces fermentations was confirmed especially in the case of L. thermotolerans. Moreover, this yeast produced high levels of the higher alcohols butanol and propanol. St. bacillaris produced significant levels of acetoin and isobutyric acid and T. delbrueckii produced an increased concentration of succinic acid. All these metabolites might play a role in maintaining intracellular redox balance. However, a more extensive systematic study is needed to investigate the extent of their involvement. The mixed cultures completed fermentation and had higher final glycerol levels than the control and lower acetic acid concentrations and therefore can contribute positively to the wine aroma. Furthermore, the mixed culture fermentations showed the potential of lowering the ethanol concentrations of wine. Furthermore it has been shown in literature that the yeasts present in the mixed culture can affect each other on gene expression level as well. However, there is little genetic information available on non-Saccharomyces yeasts. In this study, we sequenced the genes involved in glycerol and acetic acid biosynthesis of L. thermotolerans and T. delbrueckii. The gene sequences are fairly homologous with only a few differences. These gene sequences can be used to study gene expression of GPD1 and ALD6 from fermentation samples in order to determine to what extent the yeasts in a mixed culture influence the gene expression of one another.
AFRIKAANSE OPSOMMING: Druiwemos gee oorsprong aan verskeie strestoestande vir die gis wat vir alkoholiese fermentasie geïnokuleer word. Hierdie strestoestande sluit hiper-osmotiese stres, as gevolg van die hoë suiker konsentrasie, in asook redoks wanbalanse toegeskryf aan die vinnige afname in beskikbare suurstof. Tydens hierdie toestande is Saccharomyces cerevisiae geneig om gliserol as beskerming teen die osmotiese stres te produseer, sowel as vir die regenereering van reduserings ekwivalente. Die produksie van gliserol lei egter dikwels tot toenemende asynsuur produksie. Volgens literatuur kom dit voor asof menige nie-Saccharomyces giste 'n ander metabolise reaksie tot die bogenoemde stresse het, omdat daar gevind is dat hulle laer vlakke van asynsuur produseer. Eers onlangs is navorsing gedoen op die potensiële gebruik van nie-Saccharomyces giste in gemengde kulture tydens wynfermentasies. Daar is bevind dat hulle voordelige eienskappe aan die wyn kan verleen. Meeste van die nie-Saccharomyces giste lei egter tot onvolledige fermentasies soos bevesting deur hierdie studie. Dus, indien die positiewe eienskappe van hierdie giste sou benut word in wynmaak sal hulle saam met S. cerevisiae geïnokuleer moet word. Wanneer twee giste saam geïnokuleer word, beïnvloed hulle mekaar en gevolglik die wyn. In hierdie konteks was die doel van die betrokke studie om die metaboliese reaksie tot hiperosmotiese stress tydens wynfermentasies te ondersoek in die volgende wyn verwante nie-Saccharomyces giste: Lachancea thermotolerans, Torulaspora delbrueckii en Starmerella bacillaris. Fermentasies was in sintetiese druiwemos medium uitgevoer met rein kulture van die genoemde gisrasse, sowel as gemengde kulture van elke nie-Saccharomyces gis met S. cerevisiae. Die fermentasiegedarg is gemonitor en die konsentrasies van verskeie wyn verwante metaboliete is bepaal. Wat die poliol konsentrasies betref, het S. cerevisiae slegs gliserol geproduseer terwyl die nie-Saccharomyces giste additionele poliole ook geproduseer het. Die lae produksie van asynsuur in die nie-Saccharomyces fermentasies is bevestig, veral in die geval van L. thermotolerans. Verder produseer hierdie gis hoë vlakke van asetoïen en iso-bottersuur en T. delbrueckii produseer 'n hoër konsentrasie van suksiensuur. Al hierdie metaboliete mag 'n rol speel in die handhawing van intrasellulêre redoksbalans. 'n Meer uitgebreide, sistematiese studie is egter nodig om die mate van hul betrokkenheid te ondersoek. Die gemengde kulture het hul fermentasies voltooi en het hoër finale gliserol vlakke as die kontrole gehad, asook laer asynsuur konsentrasies en kan dus positief bydra tot die wyn aroma. Verder het die gemengde kultuur fermentasies die potensiaal om die etanol vlakke van wyn te verlaag, getoon. Daar is verder in die literatuur gevind dat die giste teenwoordig in die gemengde kultuur mekaar op geenuitdrukkings vlak ook kan beïnvloed. Daar is egter min genetiese inligting beskikbaar vir die nie-Saccharomyces giste. In hierdie studie het ons die gene betrokke by die produksie van gliserol en asynsuur van L. thermotolerans en T. delbrueckii se nukleotied volgordes bepaal. Die gevolglike nukleotied volgordes is redelik homoloog met net 'n paar verskille. Hierdie volgordes kan gebruik word om die geenuitdrukking van GPD1 en ALD6 vanaf fermentasie monsters te bestudeer om sodoende te bepaal tot watter mate die giste in 'n gemengde kultuur mekaar se geenuitdukking kan beïnvloed.

Mestrado em Biotecnologia
Polyhydroxyalkanoates (PHA) emerge as a promising alternative to the conventional plastics due to its biodegradability, biocompatibility and thermochemical properties. Several strategies have been studied in order to decrease PHA production costs and enlarge their commercialization, such as the use of pre-treated renewable substrates and the development of economically and environmentally sustainable extraction procedures. The first part of this work aimed to compare two different reactor operation modes for the production of short-chain organic acids (SCOA), preferred substrates for polyhydroxyalkanoates (PHA) production. Acidogenic fermentation was performed by mixed microbial cultures (MMC) in a continuously stirred tank reactor (CSTR) and in a sequential batch reactor (SBR) operated for 130 days and submitted to the same operational conditions. Both reactors achieved full fermentation (>90%) for an OLR of 10gCOD/L·d, with a different range and relative concentrations of fermentation products (FP). Ethanol (EtOH) was the dominant FP in the CSTR and acetic acid (HAc) the main SCOA produced, while the SBR presented butyric acid (HBu) as dominant FP, slightly higher than the HAc concentration. Although the CSTR showed a higher yield of SCOA than the SBR – 0.671 and 0.604, respectively -, the SBR presented a higher conversion of substrate into SCOA – 48% over 38%. The second part of the work focused on the PHA extraction from pure cultures of C. necator and MMC with surfactants and an enzymatic solution from the hydrolysate of the fungus P. variotii. An extraction protocol was developed for application of the extractive agents studied. Afterwards, a systematic extraction of PHA from the microbial cultures was performed and several concentrations and temperatures tested. Among the surfactants used, BRIJ 30 presented the most promising results with recoveries above 30% for the pure cultures and above 20% for the MMC. The enzymatic solution enabled the PHA extraction when used at a concentration of 10% but not in inferior percentages, indicating that application of higher concentrations should be studied.
Os polihidroxialcanoatos (PHA) apresentam-se como uma alternativa promissora aos plásticos convencionais, devido à sua biodegradabilidade, biocompatibilidade e propriedades termoquímicas. Várias estratégias têm sido estudadas para reduzir os custos de produção de PHA e ampliar a sua comercialização, como o uso substratos renováveis sujeitos a um pré-tratamento e o desenvolvimento de procedimentos de extração económica e ambientalmente sustentáveis. A primeira parte do trabalho consistiu na comparação de dois modos de operação de reator distintos para a produção de ácidos orgânicos de cadeia curta (AOCC), substratos preferidos para a produção de PHA. A fermentação acidogénica foi protagonizada por culturas microbianas mistas (CMM), num reator contínuo perfeitamente agitado (CSTR) e num reator descontínuo sequencial (SBR), operados durante 130 dias sob as mesmas condições operacionais. Ambos os reatores atingiram fermentação total (>90%) para a carga orgânica de 10gCOD/L·d, com gama de produtos de fermentação (PF) e respetivas concentrações relativas distintas. Etanol foi o PF dominante no CSTR e ácido acético (HAc) o principal AOCC produzido, enquanto o SBR apresentou dominância pelo ácido butírico (HBu), com concentração pouco acima do HAc. Apesar do CSTR ter tido maior rendimento de AOCC que o SBR - 0.671 e 0.604, respetivamente - o SBR teve uma conversão de substrato em AOCC superior – 48% comparativamente a 38%. A segunda parte do trabalho incidiu na extração de PHA de culturas puras de C. necator e CMM com aplicação de surfatantes e solução enzimática de hidrolisado do fungo P. variotii. Inicialmente, foi desenvolvido um protocolo para aplicação dos agentes extrativos em estudo. Após, foi feita a extração sistemática de PHA das culturas microbianas seguindo o protocolo desenvolvido e combinando valores de temperatura e concentração. Dos surfatantes utilizados, o BRIJ 30 apresentou resultados mais promissores com recuperações de polímero acima de 30% para as culturas puras e acima de 20% para as CMM. A solução enzimática permitiu extração de PHA em concentração de 10% mas não em concentrações inferiores, indicando que concentrações mais elevadas deverão ser estudadas.

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica
Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyl fatty acids, which are accumulated in microbial cells as carbon/energy reserves. PHAs are bio-based and biodegradable and display a wide range of thermoplastic properties, being a promising alternative to conventional plastics. Presently, industrial PHA production was primarily based on pure microbial cultures. Although this process has high PHA production efficiency, it presents high costs associated with the use of chemically-defined feedstocks, and to the need for sterility. An attractive feature of mixed microbial cultures (MMCs) PHAs production is the ability to use waste/surplus feedstocks. Many industrial wastes are seasonally produced making it necessary find the best method of utilization of this feedstock on PHA production process. Two different approaches might be taken account: (1) stock of industrial wastes during their production for their use throughout the year. However, the high fermentability of these agro-industrial wastes makes them susceptible to degradation during storage period; (2) the use of different feedstocks over the year according its availability. It is thus important to study MMC’s response to different feedstocks. The aim of this work is study how MMC PHA production process is affected by a feedstock shift, using cheese whey (CW) and sugar cane molasses (SCM) as model feedstocks. The use of waste based feedstock by MMCs requires a previous conversion of sugars to organic acids (OAs), which is achieved through anaerobic fermentation. In this study, a three-stage MMC PHA process was used, comprising: (1) anaerobic fermentation of surplus feedstocks to produce OAs in a membrane bioreactor (AnMBR); (2) PHA accumulating culture selection in a sequencing batch reactor (SBR) under feast and famine conditions using fermented feedstocks; and (3) PHA production using the selected cultures and the OAs produced in the earlier stages. Initially the effect of both feedstocks (CW and, SCM) in the acidogenic fermentation (stage 1) was assessed. Firstly, the AnMBR was operated under steady state with CW. When the feedstock was changed to SCM an adaption period of about 10 to 15 days was observed. When SCM was replaced by CW a faster adaptation response, approximately 7 days, was observed. The AnMBR reached similar OAs profiles in both phases when CW was fed (% g-COD HAA/g-COD OAs): 65% acetate, 10% propionate, 22% butyrate, 2% valerate, and 1% lactate. These results demonstrate that the system’s performance is reproducible. On the other hand, the anaerobic fermentation of a different feedstock, SCM, resulted in a different OAs profile (%g-COD HAA/g-COD OAs): 24% acetate, 38% propionate, 19% butyrate, and 19% valerate. In a second phase, different fermented feedstocks were used in the selection of PHA-storing organisms under a feast and famine regime in a SBR (stage 2). Initially the SBR was fed with a synthetic OAs solution; then fermented SCM (fSCM) and fermented CW (fCW) were subsequently fed as they were produced in the AnMBR. The adaption of the MMC to fSCM was faster than the adaptation to fCW. Whenever steady state was reached, PHA accumulation tests were performed using the enriched MMC fed with the corresponding feedstock (stage 3), namely synthetic OAs solution, fSCM, and fCW. Storage yields of 0.74, 0.49, and 0.73 C-mol PHA/ C-mol OAs were obtained with synthetic OAs solution, fermented molasses, and fermented CW, respectively. The culture reached a maximum PHA content of 60%, 56% and 65%, when feedstock fed were synthetic OAs solution, fSCM and fCW, respectively. A direct relation between the used feedstock and the polymers composition was observed, which was related with the different OAs profile. Even though, the shift of complex feedstock in three-stage MMC PHA process is still at a very early stage of development, this work illustrates the advantage of favoring the selection of cultures with the capacity to adapt its metabolism to different feedstocks. This will offer the possibility of using numerous substrates and improving strategies to optimize acidogenic fermentation, culture selection and polymer production.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: The quality of wine is influenced and determined by various factors, one of which includes the process of malolactic fermentation (MLF). MLF plays an integral role in the flavour and sensory profile of most red wines as well as some white wines like Chardonnay. This process is conducted by lactic acid bacteria (LAB), specifically of the genera Oenococcus, Lactobacillus, Pediococcus and Leuconostoc. Of these, Oenococcus oeni is best adapted to survive in the harsh wine environment. MLF is defined as the conversion of L-malic acid to L-lactic acid and carbon dioxide. The conversion of the dicarboxylic malic acid to the monocarboxylic lactic acid results in a decrease in acidity and an increase in pH, to give a softer mouthfeel and more favourable flavour profile. A further reason for conducting MLF in wine includes the improvement of microbial stability due to the removal of malic acid as a possible substrate for microorganisms. Recently, research focus has shifted to the ability of MLF and LAB to alter the aroma profile of wine via the production and/or modification of certain aroma compounds. In order for wine LAB to conduct MLF, they need to be able to survive the harsh and challenging wine environment. Conditions in South African wines are particularly challenging due to the long, hot ripening seasons resulting in high sugar concentrations which give high ethanol concentrations. Some LAB also struggle to adapt to an environment with high pH and low malic acid concentrations. These factors, combined with the use of sulphur dioxide, cause LAB to struggle in conducting and completing successful MLF. Many of the commercial starter cultures that are currently available contain LAB that have not been isolated from South African wine and are therefore not optimal for use under these challenging wine conditions. Oenococcus oeni is also the single LAB culture present in all commercially available starter cultures. The overriding goal of this study was to create a MLF starter culture containing a mixture of LAB cultures, namely O. oeni and Lactobacillus plantarum, which can successfully convert malic acid to lactic acid, ensure microbial stability, but also make a positive contribution to the wine aroma profile. Lactobacillus plantarum has previously been considered for possible use in a commercial starter culture. The LAB isolates used in this study were selected from the Institute for Wine Biotechnology culture collection as well as isolated from spontaneous MLF. The first objective was to characterise these LAB strains for important traits and for possible use as a MLF starter culture. A total of 23 strains were identified as O. oeni and 19 strains as Lb. plantarum. The identified strains were screened in a synthetic wine medium for their ability to convert malic acid to lactic acid. Based on the LAB strain performance in the synthetic wine medium, seven strains of both O. oeni and Lb. plantarum were selected. These 14 strains were screened for the presence of genes encoding for enzymes responsible for biogenic amine production and were found to contain none of the genes associated with the formation of histamine, tyramine or putrescine. The LAB strains were genetically screened for enzymes associated with aroma modification by LAB during MLF. The enzymes of interest that were screened for included β-glucosidase, esterase, protease and phenolic acid decarboxylase (PAD). The Lb. plantarum strains were found to possess more diverse enzymatic profiles related to aroma than O. oeni. The biggest differences were observed for the presence of β-glucosidase and PAD. The second objective was to perform small-scale fermentations with the individual LAB isolates. The individual isolates were evaluated in Pinotage and based on these results; three strains of each O. oeni and Lb. plantarum were selected for evaluation in mixed culture fermentations. The mixed cultures were evaluated in Pinotage, Shiraz and Cabernet Sauvignon in the 2008 vintage. As a third objective, the wines were also analytically and sensorially evaluated to investigate the changes in the aroma profile that could be attributed to the presence of the mixed LAB isolates. Based on the fermentation data as well as data pertaining to the aroma modification, three mixed cultures were selected for evaluation in the 2009 vintage in Pinotage, Cabernet Sauvignon and Chardonnay. The mixed cultures were able to successfully complete MLF in fermentation periods comparable to that of a commercial culture used as control. The different LAB cultures had distinct and diverse effects on the wine aroma profile. The O. oeni strain played a larger role in the ester concentration present after MLF, while the Lb. plantarum strain had a larger effect on the higher alcohol and volatile fatty acid concentration upon completion of MLF. The results generated by this novel study clearly indicate the potential of a mixed LAB starter culture for conducting MLF. The mixed cultures successfully completed MLF and made a positive contribution to the wine aroma profile.
AFRIKAANSE OPSOMMING: Die kwaliteit van wyn word beïnvloed en bepaal deur verskeie faktore en wynbereidings prosesse, wat die proses van appelmelksuurgisting (AMG) insluit. AMG speel ’n integrale rol in die sensoriese profiel van meeste rooiwyne, sowel as sommige witwyne soos Chardonnay. AMG word gedefinieër as die omskakeling van L-appelsuur na L-melksuur en koolstofdioksied. Hierdie omskakeling kan toegeskryf word aan die teenwoordigheid van melksuurbakterieë (MSB), spesifiek spesies van die genera Oenococcus, Lactobacillus, Pediococcus en Leuconostoc. Vanuit hierdie wyn MSB, is Oenococcus oeni die spesies wat die beste aanpas en oorleef onder stresvolle wyn kondisies. Die omskakeling van appelsuur, ’n dikarboksielsuur, na melksuur, ’n monokarboksielsuur, lei tot ‘n vermindering in suurheid en ’n verhoging in pH. Hierdie vermindering in suurheid gee ’n sagter en meer geronde mondgevoel aan die wyn en dra by tot ‘n meer aangename geurprofiel. ’n Verdere rede vir AMG in wyn is om mikrobiese stabiliteit te verseker deurdat appelsuur verwyder word as ’n moontlike koolstof substraat vir mikroörganismes. Onlangs het navorsing begin fokus op AMG en die vermoë van MSB om die aroma profiel van wyn te beïnvloed deur die produksie/modifisering van sekere aroma komponente. Vir MSB om AMG te kan deurvoer, moet hulle kan oorleef in die stresvolle wynomgewing. Wyntoestande in Suid-Afrika is veral uitdagend vir die oorlewing van mikroörganismes as gevolg van lang, warm somers wat lei tot ’n matriks met ’n hoë suikerkonsentrasie en wyn met ’n hoë etanolkonsentrasie. ‘n Omgewing met ‘n hoë pH en lae appelsuur konsentrasie, kan ook bydrae tot stresvolle kondisies vir MSB. Hierdie parameters, tesame met die gebruik van swaweldioksied, maak dit moeilik vir MSB om AMG te inisieer en te voltooi. Sommige van die kommersiële aanvangskulture wat tans beskikbaar is, bevat nie MSB wat onder Suid-Afrikaanse wyntoestande geïsoleer is nie en daarom is dit nie altyd optimaal vir gebruik nie. Oenococcus oeni is ook die enkele MSB kultuur wat in alle kommersiële kulture gebruik word. Die hoofdoelwit van hierdie studie was om ’n potensiële kommersiële aanvangskultuur te ontwikkel wat ‘n mengsel van MSB bevat. Hierdie aanvangskultuur moet AMG suksesvol kan voltooi, mikrobiologiese stabiliteit bevorder en steeds die wynaroma positief kan beïnvloed. Bakterierasse van O. oeni en Lb. plantarum is geselekteer vir gebruik in hierdie studie. Lactobacillus plantarum het reeds in vorige studies potensiaal getoon as ‘n moontlike aanvangskultuur. Die MSB isolate vir hierdie studie is geselekteer uit die Instituut vir Wynbiotegnologie se kultuurversameling en geïsoleer uit spontane AMG fermentasies. Die eerste doelwit was om hierdie MSB isolate te karakteriseer vir belangrike eienskappe en die moontlike gebruik as ’n kommersiële AMG aanvangskultuur. ‘n Totaal van 23 O. oeni en 19 Lb. plantarum isolate is geïdentifiseer. Hierdie isolate is in ’n sintetiese wynmedium geëvalueer vir hul vermoë om appelsuur na melksuur om te skakel. Op grond van hul reaksie in die sintetiese wynmedium, is sewe isolate van elk van die O. oeni en Lb. plantarum geselekteer. Hierdie 14 isolate is ondersoek vir die teenwoordigheid van die gene wat kodeer vir biogeenamien produksie en daar is gevind dat geen van die isolate enige van die biogeenamien gene wat ondersoek is, naamlik histamien, tiramien en putresien besit nie. Die MSB isolate is geneties ondersoek vir die teenwoordigheid van dié gene wat kodeer vir ensieme wat die aromaprofiel tydens AMG beïnvloed. Dié ensieme sluit β-glukosidase, esterase, protease, fenoliese suurdekarboksilase en sitraatliase in. Daar is gevind dat die Lb. plantarum isolate meer diverse ensiemprofiele as O. oeni besit. Die grootste verskille in die ensiemprofiele kan toegeskryf word aan die teenwoordigheid van β-glukosidase en fenoliese suurdekarboksilase. Die tweede doelwit was om kleinskaalse AMG fermentasies met die individuele MSB isolate uit te voer. Die individuele isolate is in Pinotage geëvalueer. Volgens hierdie resultate is drie isolate van elk van die O. oeni en Lb. plantarum geselekteer om in gemengde kulture getoets te word. Die gemengde kulture is in Pinotage, Shiraz en Cabernet Sauvignon in 2008 geëvalueer. As ’n derde doelwit is hierdie wyne ook analities en sensories geëvalueer om die veranderinge in die aromaprofiele as gevolg van die teenwoordigheid van die MSB te ondersoek. Op grond van die fermentasiedata, sowel as die data oor die aromaveranderinge, is drie gemengde kulture geselekteer vir evaluering in Pinotage, Cabernet Sauvignon en Chardonnay in 2009. Die gemengde kulture kon AMG suksesvol voltooi met fermentasietempo’s wat vergelykbaar was met dié van ‘n kommersiële AMG kultuur wat as kontrole gebruik is. Die verskillende MSB kulture het spesifieke en uiteenlopende uitwerkings op die wynaroma gehad. Die O. oeni isolaat in die gemengde kultuur blyk ‘n belangriker rol te speel in die esterkonsentrasie na AMG, terwyl die Lb. plantarum isolaat ’n groter effek het op die hoër alkohol en vlugtige vetsuurinhoud na AMG. Die resultate wat deur hierdie unieke studie gegenereer is, gee ’n aanduiding van die potensiaal van ’n gemengde MSB aanvangskultuur vir AMG. Die gemengde kulture kon AMG suksesvol voltooi en ‘n positiewe bydrae tot die aromaprofiel van die wyn lewer.

De nos jours, les cultures mixtes sont considérées comme une sérieuse alternative aux cultures pures pour les procédés de biotechnologie. En effet, les cultures mixtes peuvent fonctionner en réacteur continu, dans des conditions non-stériles et traiter une grande variété de substrats organiques. La principale restriction de l'utilisation de ces bioprocédés en cultures mixtes réside dans leur instabilité liée à la présence de voies métaboliques non désirées résultant d'interactions microbiennes complexes. Notamment, le rôle des bactéries de faible abondance reste à être élucidé. Ce travail a donc consisté, dans un premier temps à déterminer le rôle des bactéries minoritaires dans la production d'hydrogène par voie fermentaire en utilisant un chémostat alimenté en continu avec un milieu à base de glucose. Sept inocula ont été cultivés dans les mêmes conditions opératoires. De façon remarquable, Clostridium pasteurianum a été retrouvé comme espèce dominante de l'écosystème six fois sur sept. Seules la nature et la diversité des espèces minoritaires variaient d'un écosystème à l'autre. Ainsi, il a été montré que la structure des communautés microbiennes a une influence significative sur la production de bio-hydrogène. Au sein de ces communautés, les bactéries en proportion minoritaires jouent un rôle clé en orientant le métabolisme globale de l'écosystème. La deuxième étape de ce travail a consisté à utiliser certaines de ces espèces minoritaires comme Ingénieurs Ecologiques des Ecosystèmes microbiens (IEEM). Pour cela, la structure d'une communauté microbienne productrice d'hydrogène a été modifiée artificiellement en introduisant des souches bactériennes exogènes aux fonctions redondantes et/ou complémentaires des souches indigènes. Les résultats en réacteur batch ont montré que les performances de production d'hydrogène pouvaient être améliorées jusqu'à un facteur 3,5 par l'ajout de certaines souches. Dans l'ensemble, les résultats obtenus ne peuvent être expliqués par de simples interactions trophiques et suggèrent la présence de mécanismes d'interactions de coopération entre microorganismes. De plus, sous des conditions opératoires plus favorables (inoculum, milieu), l'insertion de certaines espèces minoritaires a permis plutôt de stabiliser le métabolisme de l'écosystème microbien sans pour autant en affecter favorablement la production d'hydrogène. Dans tous les cas, les interactions compétitives n'ont pas été favorables à la production d'hydrogène. Enfin, des essais en réacteur continu ont montré que le mode d'implantation des souches peut être un facteur primordial pour l'utilisation d'IEEM. En conclusion, ce travail a montré la potentialité d'utiliser des bactéries exogènes, en proportions minoritaires, comme facteurs biotiques pour stabiliser et/ou orienter les métabolismes microbiens vers des fonctions d'intérêt au sein des cultures mixtes microbiennes
Nowadays mixed cultures are considered as a serious alternative to pure cultures in biotechnological processes. Mixed cultures can be operated continuously, under unsterile conditions and from various organic substrates. One of the most constraints remains the chronic instability of the mixed culture processes due to the presence of unwanted metabolic pathways resulting from complex microbial interactions. More particularly the role of bacteria in low abundance remains to be elucidated. Therefore this work consisted initially to determine the contribution of sub-dominant bacteria to fermentative hydrogen production using a chemostat continuously fed with a glucose-based medium. Seven inocula were grown under the same operating conditions. Interestingly, Clostridium pasteurianum was found as dominant in six assays on seven at steady state. Only the minority bacterial population differed with regards to their identity and diversity. Acting as true keystone species, these minority bacteria impacted substantially the metabolic network of the overall ecosystem despite their low abundance. In a second step, this work consisted in using some of these minority species as Ecological Engineers of Microbial Ecosystem (EEME). In order to study this aspect, the structure of a hydrogen-producing microbial community has been artificially modified by adding exogenous bacterial strains with redundant functions and/or complementary native strains. Results in batch reactors have shown that the hydrogen production performances could be improved to a 3.5 factor by the addition of certain strains. Results obtained can not be explained by simple trophic interactions and suggest the presence of interaction mechanism of cooperation among microorganisms. Moreover, under more favourable operating conditions (inoculum, culture medium), the addition of certain species in low abundance could stabilize the metabolism of microbial ecosystem without necessarily favourably affect the hydrogen production. In all cases, competitive interactions were not favourable for hydrogen production. Trials were then realised in continuous reactors. These trials have shown that the method used to implant strains in reactors could be a key factor for using the EEME.As a conclusion, this study has shown the potential to use exogenous bacteria, in minority proportions, as biotic factors to stabilised and/or guides microbial metabolisms to functions of interest within microbial mixed cultures

Bioethanol plays a significant role in the world of liquid biofuel. However, majority of bioethanol is produced from edible food crops such as corn and sugarcane that causes an increase in demand for vacant lands for food production and, subsequently, increase in the cost of food manufacturing. Therefore, alternative raw materials for bioethanol production are sought after, such as sugarcane bagasse which is a waste material from the sugar industry. South Africa, a net sugar exporter, has a large potential to produce bioethanol from sugarcane bagasse. This research focuses on the study of the production of bioethanol from glucose and xylose which are the two most abundant sugars in hydrolysed sugarcane bagasse. To date, no suitable wild type organisms can concomitantly ferment both glucose and xylose to ethanol efficiently. Options to address the co-fermentation of glucose and xylose include genetic modification of the selected microorganism to include both pathways - limitation in the understanding of the metabolic pathways regulations - or utilization of two microorganisms in co-culture or sequential culture e.g. Zymomonas mobilis and Pichia stipitis for efficient fermentation of glucose and xylose respectively. In this study, the dual micro-organism route is explored. There are numerous problems associated with co-culturing. Xylose, a non-preferred carbon source is only converted if the glucose concentration is adequately low due to catabolite repression. In order to increase xylose conversion, a low glucose concentration is required. Therefore, two stage sequential fermentation either in one or two reactors was tested. A high inoculum of suspended or immobilized Z. mobilis was inoculated in the first stage to convert the glucose rapidly. Varying reactor configuration, including the continuous fluidized bed, continuous stirred tank reactor (CSTR) and stirred batch reactor were considered. The products and residual substrate from this fermentation was then directed to a second stage, using either a CSTR or stirred batch configuration, with a high inoculum of P. stipitis in suspension culture for conversion of xylose. When immobilized, Z. mobilis was entrapped in calcium alginate beads. On the issue of ethanol tolerance, P. stipitis is generally more easily inhibited by ethanol (threshold ethanol concentration of 35 g L-1) compared to other ethanol producing strains such as Z. mobilis (threshold ethanol concentration of 127 g L-1) and Saccharomyces cerevisiae (threshold ethanol concentration of 118.2 g L-1). In order to overcome this, a continuous bioprocess was investigated to keep ethanol concentrations in Stage II below 35 g L-1 to prevent inhibition of metabolic reactions in P. stipitis. Further, ethanol fermentation by Z. mobilis requires obligate anaerobic conditions while xylose conversion by P. stipitis is optimum under microaerobic conditions. Therefore, oxygen was sparged into the second P. stipitis stage only. The following components were carried out in this project to improve the kinetic model and to find accurate kinetic data in the selected process of the two stage sequential fermentation. Firstly, where kinetic parameters were not available in literature, the kinetic parameter relationships of glucose and xylose utilization between different constructs of the same species were examined, for example, a wild type and engineered strain. This approach was used for glucose conversion using wild type Z. mobilis, owing to the ill-fit of available kinetic parameters with experimental results. In this study, the correction factors on estimated kinetic parameters from linear and non-linear regression when a xylose fermentation route was inserted recombinantly (S. cerevisiae RWB 217) into the native culture (S. cerevisiae CEN.PK 113-7D) were determined. From kinetic parameters of an engineered strain with the xylose-fermenting pathway (Z. mobilis ZM4 (pZB5)) and the correction factors, kinetic parameters of the wild-type Z. mobilis ZM4 were determined. Predicted rates of Z. mobilis ZM4 were then validated with experimental data generated in this study. Then, the optimum initial biomass concentration required to provide a faster volumetric rate of sugar utilisation and ethanol production, as well as the optimum oxygenation level for xylose conversion using P. stipitis achieved through appropriate aeration were investigated through experimental observation and using a MATLAB mathematical model developed through combination of the Andrews and Levenspiel's models, with oxygen, substrate, cell and product terms. Experiments were carried out to validate the kinetic model and data under anaerobic and microaerobic growth conditions in a batch process. The results showed that both increasing the initial biomass concentration (3 g L-1) and operating under optimum oxygenation levels (0.1 vvm) benefitted the ethanol production and yield by P. stipitis from xylose. It was also concluded that the addition of the oxygen effective factors in the developed model allowed for optimization of aeration in the fermentation system. Next, the custom kinetic model for fermentation process of bioethanol production was developed in Aspen Custom Modeller (ACM) and embedded in Aspen Plus. The model includes equations of vapour-liquid equilibrium (VLE), mass balance, and energy balance (e.g. molecular weight, thermodynamic phase equilibria, kinetic equation). The obtained results showed better agreement between industrial data and kinetic model (1% differences) than a stoichiometric model (9% differences). The simulation showed that ACM integrated into Aspen Plus allowed for complex biological processes to be accurately predicted for biomass growth, ethanol production and sugar consumption. Finding suitable microorganisms and process conditions for efficient glucose and xylose conversion is still currently a challenge and requires optimization. Therefore, this research focusses on improving the conversion of glucose and xylose to bioethanol, with specific emphasis on the fermentation systems used to maximize biomass efficiency, and ethanol yields and productivities. Manipulation of process conditions ranging from operation conditions (e.g. batch, fed-batch, continuous), process parameters (aeration, temperature, pH), immobilization technique and type of microorganism initially using kinetic models and thereafter validating with experimental data, therefore, offers a quick and strong foundation in improving bioethanol yields and productivities.

Cette étude vise à étudier l'effet de la composition de substrats organiques solides sur les performances de production d'hydrogène, les voies métaboliques associées et les changements des communautés microbiennes dans un réacteur discontinu (sCSTR). L'hydrogène est un vecteur énergétique idéal qui a gagné en intérêt scientifique au cours de la dernière décennie. L'H2 produit par voie biologique, ou biohydrogène, peut être produit par des procédés de fermentation sombre où les déchets organiques sont traités et avec la production de molécules à haute valeur ajoutée. Cependant, l'effet de la composition des déchets organiques solides sur la production de biohydrogène dans la fermentation sombre n'a pas encore été clairement élucidé. Au cours de cette étude, une revue bibliographique a été réalisée sur la production d'hydrogène à partir de déchets agricoles. Cette revue montre qu'une large gamme de performances en hydrogène peut être observée principalement en raison de la variabilité dans les compositions en même type de substrats et des conditions expérimentales appliquées. Après avoir optimisé un protocole de test de potentiel biohydrogène (BHP), une grande variété de substrats organiques solides visant à couvrir un grand panel de déchets a été testée pour fournir des données comparables à analyser. Les résultats d'une régression PLS ont montré que seuls les sucres solubles ou facilement disponibles éteint corrélaient avec la production d'hydrogène. En outre, les rendements d'hydrogène corrélaient aussi bien avec l'accumulation de butyrate, principale voie productrice de bioH2. Un modèle prédictif du rendement en hydrogène en fonction de la teneur en sucres a été proposé. Ensuite, des expériences ont été menées en réacteur semi-continu (sCSTR) avec le topinambour comme substrat solide. Il a été montré qu'une faible charge organique favorisait une production continue d'hydrogène tandis que l'accroissement de la charge organique introduisait la présence de voies concurrentes à la production d'hydrogène. De plus, les profils des empreintes moléculaires basées sur l'ADNr 16s ont montré que l'augmentation de la charge organique avait un impact significatif sur la diversité microbienne en favorisant l'implantation de microorganismes ne produisant pas d'hydrogène tels que des bactéries lactiques
This study aims to investigate the effect of solid substrates composition on hydrogen production performances, metabolic pathways and microbial community changes in batch reactor and their dynamics in semi continuous reactors (sCSTR). Hydrogen is an ideal energy carrier which has gained scientific interest over the past decade. Biological H2, so-called biohydrogen, can especially be produced by dark fermentation processes concomitantly with value-added molecules (i.e. metabolic end-products), while organic waste is treated. However, the effect of solid organic waste composition on biohydrogen production in dark fermentation has not yet been clearly elucidated. In this study, a bibliographic review was made on hydrogen production from agricultural waste. This survey on literature showed that diverse performances were reported on hydrogen production due to the variability in substrate compositions and experimental conditions. After having optimized a protocol of biohydrogen potential test (BHP), a wide variety of organic solid substrates aiming to covering a large range of solid waste was tested to provide a comparable data analysis. The results of a PLS regression showed that only soluble carbohydrates or easily available carbohydrates correlated with hydrogen production. Furthermore, hydrogen yields correlated as well with butyrate H2-producing pathway which is consistent with the literature knowledge. A predictive model of hydrogen yield according to carbohydrate content was proposed. Then, experiments were carried out in sCSTR with Jerusalem artichoke tubers as a case study. It was shown that low organic loading rate favored continuous hydrogen production while higher organic loading introduced hydrogen competition pathways and decreased the overall hydrogen yields. Moereover, 16S rRNA gene based CE-SSCP profiles showed that increasing OLR had a significant effect on the microbial diversity by favoring the implementation of microorganisms not producing hydrogen, i.e. lactic acid bacteria

L'hydrogène, qui constitue une solution alternative et durable à l’usage d’énergies fossiles, est produit essentiellement par reformage de combustibles fossiles (95%). Des filières de production plus soucieuses de l'environnement sont envisagées. Deux familles de technologies sont explorées: 1) par décomposition thermochimique ou électrochimique de l'eau et 2) à partir de différentes sources de biomasse. Parmi celles-ci, les cellules d'électrolyse microbienne ou «Microbial electrolysis cell (MEC)» permettent de produire de l'hydrogène par électrolyse de la matière organique. Une MEC consiste en une cathode classique qui assure la production d'hydrogène par la réduction électrochimique de l'eau, associée à une bioanode qui oxyde des substrats organiques en dioxyde de carbone. Ce processus d'oxydation n'est possible que grâce au développement sur l'anode d'un biofilm microbien électroactif qui joue le rôle d'électro-catalyseur. Par rapport aux procédés courants d'électrolyse de l'eau, une MEC requière un apport énergétique 5 à 10 fois plus faibles. En outre, les procédés « classiques » de production de bio-hydrogène par voie fermentaire en cultures mixtes convertissent des sucres avec des rendements limités à 2-3 moles d'hydrogène par mole d'hexose tout en coproduisant des acides organiques. Alimenté par de l'acétate, une MEC produit au maximum 3 moles d'hydrogène/mole d'acétate. Le couplage de la fermentation à un procédé d'électrolyse microbienne pourrait donc produire de 8 à 9 moles d'hydrogène/mole d'hexose, soit un grand pas vers la limite théorique de 12 moles d'hydrogène/mole d'hexose. L'objectif de cette thèse est d'analyser les liens entre la structure des communautés microbiennes dans les biofilms électroactifs et en fermentation, les individus qui les composent et les fonctions macroscopiques (électroactivité du biofilm, production d'hydrogène) qui leur sont associées dans des conditions permettant de réaliser le couplage des deux procédés. L'originalité de cette étude a été de travailler en milieu salin (30-35 gNaCl/L), favorable au transport de charges dans l'électrolyte de la MEC. Dans un premier temps, la faisabilité de la fermentation en conditions salines (3-75 gNaCl/L) a été démontrée en lien avec l'inhibition de la consommation de l'hydrogène produit et une forte prédominance d'une nouvelle souche de Vibrionaceae à des concentrations en sel supérieures à 58 gNaCl/L. D'autre part, la mise en œuvre de biofilms électroactifs dans des conditions compatibles avec la fermentation sombre a permis la sélection d'espèces dominantes dans les biofilms anodiques et présentant des propriétés électroactives très prometteuses (Geoalkalibacter subterraneus et Desulfuromonas acetoxidans) jusqu'à 8,5 A/m². En parallèle, la sélection microbienne opérée lors d'une méthode d'enrichissement utilisée pour sélectionner ces espèces à partir d'une source d'inoculum naturelle sur leur capacité à transférer leurs électrons à des oxydes de Fer(III) a été étudiée. Une baisse des performances électroactives du biofilm liée à une divergence de sélection microbienne dans ces deux techniques de sélection mène à limiter le nombre de cycle d'enrichissement sur Fer(III). Cependant, l'enrichissement sur Fer(III) reste une alternative efficace de pré-selection d'espèces électroactives qui permet une augmentation de rendement faradique de 30±4% à 99±8% par rapport au biofilm obtenu avec un inoculum non pré-acclimaté. Enfin, l'ajout d'espèces exogènes issues de la fermentation sombre sur le biofilm électroactif a révélé une baisse de l'électroactivité du biofilm se traduisant par une diminution de la densité de courant maximale produite. Cette baisse pourrait s'expliquer par à une diminution de la vitesse de transfert du substrat due à un épaississement apparent du biofilm. Cependant, un maintien de sa composition microbienne et de la quantité de biomasse laisse supposer une production d'exopolymères (EPS) dans le biofilm en situation de couplage
Nowadays, alternative and sustainable solutions are proposed to avoid the use of fossil fuel. Hydrogen, which constitutes a promising energy vector, is essentially produced by fossil fuel reforming (95%). Environmentally friendly production systems have to be studied. Two main families of technologies are explored to produce hydrogen: 1) by thermochemical and electrochemical decomposition of water and 2) from different biomass sources. Among those last ones, microbial electrolysis cells (MEC) allow to produce hydrogen by electrolysis of organic matter. A MEC consists in a classical cathode, which provides hydrogen production by electrochemical reduction of water, associated to a bio-anode that oxidizes organic substrates into carbon dioxide. This process is only possible because of the anodic development of an electroactive microbial biofilm which constitutes an electrocatalyst. In comparison to classical water electrolysis process, a MEC requires 5 to 10 times less electrical energy and therefore reduces the energetic cost of produced hydrogen. Furthermore, classical process of dark fermentation in mixed cultures converts sugars (saccharose, glucose) to hydrogen with a limited yield of 2-3 moles of hydrogen per mole of hexose because of the coproduction of organic acids (mainly acetic and butyric acids). Fed with acetate, a MEC can produce up-to 3 moles of hydrogen per mole of acetate. Therefore, the association of these two processes could permit to produce 8 to 9 moles of hydrogen per mole of hexose, which represents a major step toward the theoretical limit of 12 moles of hydrogen per mole of hexose.Therefore, this work aims at analyzing the relationship between microbial community structures and compositions and the associated macroscopic functions (biofilm electroactive properties, hydrogen production potential) in electroactive biofilms and in dark fermentation in conditions allowing the coupling of the two processes. The originality of this study is to work in saline conditions (30-35 gNaCl/L), which favors the charges transfer in the MEC electrolyte.First of all, feasibility of dark fermentation in saline conditions (3-75 gNaCl/L) has been shown. This was linked to an inhibition of produced hydrogen consumption and the predominance of a new Vibrionaceae species at salt concentrations higher than 58 gNaCl/L. Secondly, electroactive biofilm growth in conditions compatibles to dark fermentation (pH 5.5-7 and fed with different organic acids) allowed to select dominant microbial species in anodic biofilms that present promising electroactive properties (Geoalkalibacter subterraneus and Desulfuromonas acetoxidans) with maximum current densities up to 8.5 A/m². In parallel, the microbial selection occurring during iron-reducing enrichment method used to select species from a natural inoculum source and based on their capacity to transfer electrons to iron oxydes (Fe(III)) has been studied. A decrease of electroactive performances of the biofilm linked to the divergence of microbial selection led to a limitation of the number of iron-enrichment steps. However, enrichment on Fe(III) presents an efficient alternative to pre-select electroactive species with an increase of coulombic efficiency from 30±4% to 99±8% in comparison with a biofilm obtained with a non-acclimated inoculum. Finally, the addition of exogenous bacteria from a dark fermenter on the electroactive biofilm revealed a decrease of electroactivity with a decrease of maximum current density produced. This diminution could be explained by a lower substrate transfer due to an apparent thickening of the biofilm. Nevertheless, the stability of microbial composition and of bacterial quantity on the anode suggests that a production of exopolymers (EPS) occurred

Mestrado em Biotecnologia
The acidogenic fermentation is the second phase of anaerobic digestion. Volatile fatty acids (VFAs) are the main products of acidogenic fermentation and can act as substrates for hydrogen or polyhydroxyalkanoates (PHA) production. In this work, mixed microbial cultures (MMC) collected in a wastewater treatment plants were used for acidogenic fermentation of hardwood spent sulphite liquor (HSSL), a by-product of paper and pulp industry, in a continuously stirred tank reactor (CSTR), and cheese whey permeate (CWP) in a sequential batch reactor (SBR) in non-sterile conditions. In HSSL fermentation, HRT of 1.6 and 2 days were tested with a feeding concentration of 15 g COD/L. pH was left uncontrolled, but was stable around 5.0 ± 0.24 due to the buffer effect of HSSL. The highest conversion of 10 %, with 68% of sugars, and degree of acidification of 10 % were obtained at HRT 2 days and OLR of 7.7 g COD/(L·d). By using CWP a high conversion of acidogenic fermentation (> 80 %) was achieved, even at a pH below the optimal value (4.5). pH and HRT influenced sugars to VFA conversion and the profile of products. Conversion improved with pH and HRT and ethanol-type and propionic-type fermentation were identified at different pHs. Finally from the conditions tested for acidogenic fermentation of CWP, pH 4.5, HRT 24 h and 30 ºC led to the reduction of base consumption at high conversion.
A fermentação acidogénica é a segunda fase da digestão anaeróbica. Ácidos gordos voláteis (AOVs) são os principais produtos da fermentação acidogénica e podem servir como substratos na produção de hidrogénio ou polihidroxialcanoatos (PHA). Neste trabalho, culturas microbianas mistas (MMC) recolhidas numa estação de tratamento de águas residuais foram usadas para acidificar alguns componentes do licor de cozimento ao sulfito ácido (HSSL), um subproduto da indústria papeleira, num reator contínuo perfeitamente agitado (CSTR), e permeado de soro de queijo (CWP) num reator descontínuo sequencial (SBR), em condições não estéreis. Na fermentação acidogénica do HSSL, foi testado o efeito de HRTs de 1,6 e 2 dias com uma alimentação de 15 g COD/L, com 22 % de glucose e xilose. O pH não foi controlado, mas manteve-se estável em cerca de 5,0 ± 0,24 devido ao efeito tampão do HSSL. A maior conversão e grau de acidificação obtidos foram ambos de 10% com uma HRT de 2 dias e OLR de 7,7 g de COD/(L·d). O consumo de açúcares foi de 68 %. Na fermentação de CWP foi atingida uma conversão elevada (> 80%), mesmo com pH controlado a um valor inferior ao ótimo (4,5). O pH e o HRT influenciaram a conversão de açúcares em VFA e o perfil de produtos. A conversão melhorou com o pH e a HRT e fermentação do tipo etanólico e propiónico foram identificadas a pHs diferentes. Verificou-se que o conjunto de condições testadas que conduziu a uma redução do consumo de base, com elevada conversão, foi pH 4,5, HRT 24 h e 30 ºC.

La Fermentation Malo-Lactique (FML) réalisée par OEnococcus oeni est une étape importante de la vinification qui doit être maîtrisée. Bien que les vinificateurs aient à leur disposition des souches OE. oeni selectionnees la FML n’est pas toujours réussie. Les conditions physico-chimiques (pH, éthanol, température), la composition du vin et les facteurs biologiques influencent l’activite de cette bacterie ; parmi ces dernières les interactions entre micro-organismes sont primordiales. Souvent, après la fermentation alcoolique par la levure, des souches indigenes d’OE. oeni sont naturellement présentes dans le vin. Des interactions négatives peuvent alors se produire entre les souches autochtones et les souches sélectionnées apportées. Des connaissances sur ces interactions sont donc necessaires. L’objectif de ce travail etait d’etudier les interactions pendant la FML entre 5 souches d’OEnococcus oeni issues de différentes niches écologiques. Pour cela, des expériences ont été effectuées dans du milieu MRS modifié et dans des conditions proches à celles du vin (20 °C ; pH 3,5 et 10 % d’ethanol). Nous avons tout d’abord caracterise le comportement des souches en cultures pures à la fois dans les conditions de micro-aerobie et d’anaerobiose. Une grande variabilité a été retrouvée entre les souches dans les 2 conditions : trois des 5 souches sont favorisées en conditions d’anaerobiose tandis que les deux autres se sont mieux développées en conditions de micro-aérobie. La présence de 4 g.L-1 d’acide L-malique dans le milieu permet de produire, pour toutes les souches, une biomasse environ 2 fois plus élevée que celle obtenue dans le milieu sans acide L-malique. La totalite de l’acide malique est consommee par les 5 souches mais avec des vitesses différentes. Pour une souche donnée la vitesse spécifique de consommation d’acide L-malique (ν) et la vitesse specifique de croissance (μ) presentent des profils similaires au cours de la FML. Elles ont été reliées par un modèle mathématique qui a permis de quantifier ce lien pour chaque souche. Les interactions lors des cultures mixtes des 10 couples formés par les 5 souches ont ensuite été étudiées dans un Bio-Réacteur à Membrane (BRM) en anaérobiose. Trois catégories ont été mises en évidence: interactions à effets négatifs réciproques sur la croissance des 2 souches en culture mixte ; interactions à effet négatif sur la croissance de la souche la plus rapide en culture pure et à effet positif sur la croissance de la souche la plus lente en culture pure et interactions à effets positifs sur la souche la plus rapide en culture pure. La comparaison des cultures pures et mixtes a révélé que l’activite specifique de croissance des souches est affectee en culture mixte, ce qui provoque le prolongement de la phase de la latence dans le cas de l’inhibition et son raccourcissement dans le cas de la stimulation. La modelisation de la consommation d’acide L-malique a révélé pour certains couples une activation de la consommation de cet acide bien que la croissance soit fortement inhibée. Ces interactions, qui affectent le déroulement de la FML, ne peuvent etre dues qu’a l’effet de metabolite(s) extracellulaire(s) excretee(s) dans le milieu de fermentation. Ces métabolites restent à identifier
In winemaking, the control of malolactic fermentation (MLF) by OEnococcus oeni is an essential step for this process. Although winemakers have the availability for selected OE.oeni strains, the MLF is not always successful. The physical-chemical conditions (pH, ethanol, and temperature), the composition of wine, and biological factors, all together influence the activity of this bacterium; regarding biological factors, the interactions between microorganisms are essential. Often, after alcoholic fermentation by yeast, indigenous strains of OE.oeni are naturally present in wine, negative interactions can then occur between the indigenous strains and selected strains; therefore, knowledge on these interactions is needed. The goal of the present work was to study the interactions during MLF between five strains of OE.oeni from different origins. Experiments were performed in the modified MRS medium to be in nearly conditions to those of wine (20 °C, pH 3.5, and 10% ethanol). The characterization of the behavior of strains in pure cultures was done under both, micro-aerobic and anaerobic conditions; a large variability was found between the strains in the two conditions: three out of five strains were favored under anaerobic conditions while the two others were better developed in micro-aerobic conditions. The presence of 4 g.L-1 of L-malic acid in the culture medium increased the biomass produced, about two-fold higher than that obtained in medium without L-malic acid. All of the L-malic acid is consumed by the five strains but at different specific rates. A mathematical model allowed to quantifying the relationship between the specific consumption rate of L-malic acid (ν) and the specific growth rate two specific rates for each strain; for a given strain, both rates have similar profiles during the MLF. Interactions in mixed cultures of 10 couples formed by the five strains were then examined in a Membrane Bioreactor (BRM) under anaerobic conditions. Three different interaction types were identified: 1) negative reciprocal interactions of the both strains in mixture culture, 2) interaction that affect negatively the favored strain in pure culture and positively the slowest one, and 3) interaction with positive effect on the fastest strain in pure culture. Comparison of pure and mixed culture showed that the specific activity of strains was affected in mixture culture causing the extension of the lag phase in the case of inhibition and its shortcut in the case of stimulation. Modeling of the consumption of the L-malic acid revealed activation of the consumption of this acid for some couples however, growth is strongly affected. The interactions affecting the course of the MLF are due solely to the effect of excreted extracellular metabolite(s); these metabolites remain to be identified

Dans le contexte du développement de l’hydrogène-énergie, de nouvelles voies de production renouvelables sont étudiées, parmi lesquelles la fermentation obscure est un processus biologique convertissant la biomasse. Dans cette étude, ce procédé a été optimisé en réacteur agité semibatch par la sélection de cultures mixtes (boues de station d’épuration) et l’optimisation des paramètres de fermentation associés (température, ajout de substrat, régulation du pH). La présence majoritaire de bactéries du genre Clostridium a été observée dans le milieu fermentaire. Différents modes d’extraction des gaz produits ont été évalués, permettant d’intensifier le procédé par l’utilisation d’un gaz de balayage (N2 ou CO2). La mise en œuvre efficace en fonctionnement continu d’un bioréacteur membranaire dans une configuration d’extraction gaz/liquide a permis d’améliorer le rendement (> + 90%) et la productivité en H2 (> + 300%) par rapport au mode de fonctionnement continu en réacteur agité. Enfin, l’utilisation d’un substrat réel (bourbes viticoles) a permis de prouver la faisabilité du procédé dans une perspective d’industrialisation
In the context of the development of hydrogen-energy, new renewable production ways are studied, among which dark fermentation is a biological process converting the biomass. In this study, this process was optimized for a semibatch reactor by the selection of mixed cultures (waste water treatment plant sludges) and the optimization of associated parameters of fermentation (temperature, add of substrate, pH regulation). The presence in majority of bacteria from the genus Clostridium was observed in the fermentation broth. Different extraction modes of the produced gas were evaluated, allowing to intensify the process by the use of a sparging gas (N2 or CO2). The successful implementation in continuous mode of a membrane bioreactor in a configuration of gas/liquid extraction allowed an increase in H2 yield (> + 90%) and productivity (> + 300%) compared to the continuous stirred tank reactor. Finally, the use of a real substrate (winery waste) allowed to prove the feasibility of this process in the prospect of industrialization

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: The use of non-Saccharomyces yeasts together with Saccharomyces cerevisiae in mixed starter cultures has become an accepted oenological tool to enhance the organoleptic properties of wine. Recent studies have indeed demonstrated the positive contribution that non- Saccharomyces yeasts may have on the bouquet of wine. These mixed starter cultures are characterized by high inoculation levels of individual strains into the must, and each strain in turn is characterized by its own specific metabolic activity. These factors lead to a multitude of interactions occurring between the individual populations within the must. The fundamental mechanisms which drive these interactions are still largely unknown, but several studies have been conducted in order to investigate the metabolic outcome of these interactions. In this study, we endeavour to further characterize the interactions which occur between four individual non-Saccharomyces yeast strains in mixed culture fermentation with S. cerevisiae. Metschnikowia pulcherrima IWBT Y1337, Lachancea thermotolerans IWBT Y1240, Issatchenkia orientalis Y1161 and Torulaspora delbrueckii CRBO LO544 were used in mixed culture fermentations with a commercial strain of S. cerevisiae at an inoculation ratio of 10:1 (non-Saccharomyces: S. cerevisiae). The biomass evolution and fermentation kinetics of both participating species were affected by the high cell density of the other, with neither population reaching the maximal density attained by the pure culture fermentation. The final wine composition of each individual mixed fermentation showed clear differences, from the pure cultured S. cerevisiae and from each other, based on the concentrations of the major volatile compounds found in the wine. Upon further characterization of these specific mixed culture fermentations, it was found that each individual combination of non-Saccharomyces and S. cerevisiae produced similar increases and decreases of certain major volatile compounds as demonstrated by previous authors, using the same combination of non-Saccharomyces species together with S. cerevisiae. From a winemaking perspective, the use of these non- Saccharomyces yeast strains in combination with S. cerevisiae could be a useful strategy to diversify the chemical composition of wine, by increasing the concentration of certain desirable volatile compounds and by modulating the concentration of undesirable metabolites. Furthermore, this research serves as a foundation for further elucidation of the interactions which drive these metabolic outcomes in response to the high cell density of two yeast populations in mixed culture fermentations.

L'utilisation de souches de levures sélectionnées pour réaliser la FA est une pratique très répandue en œnologie. Après le développement de l'utilisation de levains de souche pure de Saccharomyces, l'innovation est aujourd'hui dans la mise en œuvre de levains mixtes de Saccharomyces et de non-Saccharomyces qui permettent de diversifier les produits finaux obtenus. La problématique réside dans l'existence d'interactions entre les souches rendant difficile la maîtrise de la fermentation. T. delbrueckii présente dans la flore indigène du mout de raisin est une des levures non-Saccharomyces les plus appropriées pour entrer dans la composition de ces levains mixtes. En effet, elle présente une bonne capacité fermentaire et peut permettre d'augmenter la complexité aromatique du vin ou encore de réduire son acidité volatile. L'objectif de ce travail était d'étudier les interactions pendant la FA entre des souches sélectionnées pour l'oenologie : une T. delbrueckii et une S. cerevisiae. Pour cela des expériences ont été réalisées dans des milieux synthétiques simulant le moût de raisins blancs. Le comportement des souches pures a tout d'abord été caractérisé. Il a été montré que la souche S. cerevisiae avait de meilleures performances fermentaires d'un point de vue cinétique que la souche T. delbrueckii. Toutefois, T. delbrueckii a montré des capacités acceptables pour épuiser les sucres et surtout a permis d'obtenir des profils aromatiques différents. Le comportement vis-à-vis de l'oxygénation des moûts de ces deux levures est assez semblable, T. delbrueckii étant cependant beaucoup plus sensible à ce paramètre que S. cerevisiae. L'interaction entre ces deux levures a ensuite était étudiée dans un bioréacteur à membrane sous anaérobie stricte dans différentes conditions : composition en azote assimilable du milieu et stratégie d'inoculation. Il a été clairement mis en évidence que T. delbrueckii était affectée par la présence de S. cerevisiae. Le type d'interaction soupçonné est celui d'amensalisme lié à l'excrétion par S. cerevisiae d'un constituant toxique pour T. delbrueckii. Dans ces conditions, la stratégie d'inoculation recommandée est l'ensemencement séquentiel des levures : T. delbrueckii en début de fermentation, puis l'ajout de S. cerevisiae 48 h après. Ceci permet à T. delbrueckii de se développer et d'exprimer son potentiel aromatique avant que S. cerevisiae ne soit introduit pour assurer une fin rapide de la fermentation. Toutefois, nous avons montré que même dans ces conditions, l'implantation de T. delbrueckii n'était pas garantie car, le moût n'étant pas stérile, une présence, même faible, de S. cerevisiae dans la flore naturelle peut inhiber sa croissance. Par ailleurs, il a été mis en évidence que dans les mouts à faible teneur en azote initial, ce constituant pouvait être épuisé au moment de l'inoculation de S. cerevisiae. Dans ces conditions, S. cerevisiae ne peut se développer et l'achèvement de la fermentation est alors problématique
The use of the selected yeast strains to realize the alcoholic fermentation is very prevalent practice in vinification. After the development of utilization of the preparation of pure Saccharomyces cerevisiae strain, the innovation is now to apply the mixte starter cultures of Saccharomyces and non-Saccharomyces that allow to diversifying the obtained final products. The problem resides in the existence of interactions between the strains giving the difficulty to controle the fermentation. Torulaspora delbrueckii present in the indigenous flora of the grape must is one of the most appropriate non-Saccharomyces yeasts to enter in the composition of these multistater cultures. In fact, this strain has been presented a good fermentative capacity and could allow to increasing not only the aromatic complexity of wine, but also to reducing its volatile acidity. The objective of our work is to study the interactions during the alcoholic fermentation between the selected strains for enology: one T. delbrueckii and one S. cerevisiae. For this reason, the experiments were realized in the synthetic mediums simulated to the white grape must. The behaviours of the pure strains were firstly characterized. It was shown that the S. cerevisiae strain had the best fermentative performances, a critical point in comparaison with the T. delbrueckii strain. Nevetheless, T. delbrueckii showed the acceptable capacities to exhaust the sugars and especially to allow us to obtain the different aromatic profiles to that of S. cerevisiae. The behaviour via the oxygenation to the musts of these two yeasts is enough close, T. delbrueckii being however much more sensible to that parameter than . cerevisiae. The interactions between these two yeasts were then studied in a membrane bioreacteur under strict anaerobie in different conditions: composition in assimilable nitrogen of the medium and strategy of inoculation. It has been clearly demonstrated that T. delbrueckii has been affected by the presence of S. cerevisiae. The suspected type of this interaction is the amensalism one bound to a toxic compound excreted by S. cerevisiae. In these conditions, the recommended inoculation strategy is the sequential culture of these yeasts: T. delbrueckii at the beginning of the fermentation, then the addition of S. cerevisiae after 48 h. This allows T. delbrueckii to develop and express its potentiel of aromatic production before the S. cerevisiae is introduced to assure a rapid finish of the fermentation. However, we showed that even in these conditions, T. delbrueckii growth has been not guaranteed because of, since the must is not sterilized, a presence even small of S. cerevisiae in the natural flore can inhibite the croissance of the former. It has been also demonstrated that in the must with low intitial nitrogen content, this compound could be exhausted at the moment of the S. cerevisiae inoculation. In these conditions, S. cerevisiae can not develop and the achievement of the fermentation is yet problematic

Mestrado em Biotecnologia
The excessively use of fossil fuels led to the necessity of more sustainable processes using renewable resources, such as biomass. Due to its abundance, low cost and broad availability, lignocellulosic biomass is a promising substrate for the production of chemicals and energy, among others. Short-chain organic acids (SCOA) have a great potential not only due to their wide applicability, but also to the fact that they can be produced biologically through acidogenic fermentation (AF) from renewable resources, such hardwood sulfite spent liquor (HSSL). The optimization and control of this process is crucial and comprises the monitoring of parameters such as temperature, pH, organic loading rate, sludge and hydraulic retention times, origin of the inoculum and reactor type. In the present study, the AF of HSSL was evaluated in a continuous stirred tank reactor (CSTR), without (CSTR1) and with pH control (CSTR2). For CSTR1, two retention times were tested, 2.34 and 3.01 days, being the average SCOA concentrations of 3.10 and 3.53 gCOD/L achieved. For CSTR2, three pH values were tested, pH 6, 7 and 8. The average SCOA concentrations achieved were 2.36, 2.38 and 2.27 gCOD/L, respectively. Lastly, a moving bed biofilm reactor (MBBR) was also tested and an average concentration of 2.71 gCOD/L of SCOA was obtained. Generally, the main SCOA produced were acetic, propionic and butyric acids. The batch experiments performed, plus the results from all the reactors, allowed to conclude that pH present a decisive influence on the SCOA profiles achieved.
O uso excessivo dos recursos fósseis está na origem da introdução de processos mais sustentáveis, que usam fontes renováveis como matéria prima, como a biomassa. Devido à sua abundância, baixo custo e vasta disponibilidade, a biomassa lenhocelulósica apresenta-se como um substrato promissor para a produção de químicos e energia, entre outros. Os ácidos orgânicos de cadeia curta (SCOA) apresentam um enorme potencial devido à sua ampla aplicabilidade e ao facto de poderem ser produzidos biologicamente, por fermentação acidogénica (AF), a partir de fontes renováveis, como é o caso do licor de cozimento ao sulfito ácido (HSSL). A otimização e controlo deste processo é crucial e inclui a monitorização de parâmetros como a temperatura, pH, taxa de carga orgânica, tempo de retenção, origem do inóculo e conformação do reator. No presente trabalho, a AF do HSSL foi avaliada num reator contínuo com mistura perfeita (CSTR), sem (CSTR1) e com controlo de pH (CSTR2). Para o CSTR1, dois tempos de retenção foram testados, 2,34 e 3 dias, tendo sido obtidas as concentrações médias de 3,10 e 3,53 gCOD/L de SCOA. Para o CSTR2, foram testados os valores de pH 6, 7 e 8, tendo sidas obtidas as concentrações médias de 2,36, 2,38 e 2,27 gCOD/L de SCOA. Por último, foi também testado um reator de biofilme de leito móvel (MBBR), tendo sido obtida uma concentração média de 2,71 gCOD/L de SCOA. De uma forma geral, os SCOA maioritariamente produzidos foram os ácidos acético, propiónico e butírico. Os testes batch realizados, juntamente com o CSTR2, permitiram ainda concluir que o pH tem uma influência decisiva nos perfis de SCOA obtidos.

The pursuit of flavour and phenolic ripeness, augmented by climate warming and extreme weather events, often leads to excessive sugar accumulation in grapes translating to undesirably high ethanol content in wines. Other common characteristics of such grapes and wines are inadequate acidity and aroma profiles, which all together compromise the quality and marketability of the final product. To tackle these issues, research has devised a number of methods implemented across the entire grape and wine production chain. Among these, partial fermentations with non-Saccharomyces yeasts is of particular interest, as it represents an undemanding approach that can also impart ‘complexity’ and distinctness. However, the full potential of non-Saccharomyces yeasts in wine ethanol and flavour management remains elusive, and this work aimed to further explore it. Research has shown that mixed fermentations with non-Saccharomyces yeasts can lead to enhanced wine aroma and sensory properties, and albeit limited, the current range of non- Saccharomyces is indeed marketed for this purpose. The potential of commercially available (and thus readily implementable) non-Saccharomyces co-inocula was assessed in Shiraz fermentations at two maturity levels; earlier harvest (24 °Brix) and technical ripeness (29 °Brix). Eight yeast treatments trialled in pilot scale fermentations included sequential inoculations using three Torulaspora delbureckii strains, one strain each of Lachancea thermotolerans and Metschnikowia pulcherrima, a commercial blend of non-Saccharomyces and S. cerevisiae, and appropriate S. cerevisiae controls. Fermentation monitoring and comprehensive chemical and sensory analysis allowed for the comparison of the treatments. The results showed a pronounced matrix-derived modulation of wine profiles which was reflective of grape maturity levels. Within each harvest date, however, the yeast treatments had a significant impact on a range of compositional and wine sensory characters. At earlier harvest stage, certain non-Saccharomyces treatments, in particular T. delbrueckii, led to an increased wine sensory appeal (i.e. ‘floral’, ‘red fruit’, ‘aroma intensity’, ‘spice’) compared to the S. cerevisiae control (‘vegetal, ‘acidic’ and ‘bitter’). These treatments, however, were related to incomplete fermentations in higher ripeness conditions. Thus, some non-Saccharomyces yeast showed promise in enhancing the quality of wines produced from earlier harvested grapes and as such represent a complementary approach in managing wine ethanol concentrations. The following study addressed the selection of lower-ethanol producing non-Saccharomyces strain(s) for use in sequential cultures with Saccharomyces cerevisiae. Oenological performances of six M. pulcherrima strains were evaluated in fermentations with S. cerevisiae inoculated after seven days. The best-performing M. pulcherrima MP2 strain was further characterised in six sequential fermentations with different S. cerevisiae inoculation delays in both synthetic and white grape juice. The analysis of main metabolites, undertaken prior to sequential inoculations and upon fermentation completion, highlighted metabolic interactions and carbon sinks other than ethanol in mixed culture fermentations. Depending on the inoculation delay, MP2 white wines contained between 0.6% and 1.2% (v/v) less ethanol than the S. cerevisiae control, with even larger decreases achieved in the synthetic juice. The MP2 wines also had higher concentrations of glycerol and lower concentrations of acetic acid. The analysis of volatile compounds revealed compositional alterations arising from the S. cerevisiae inoculation delay, with increased acetate esters and higher alcohols detected in all analysed MP2 treatments. The concept of intra-specific variability was studied using L. thermotolerans as a model. This species harbours several metabolic traits that are of value in oenology, including lactic acid production, potential to decrease ethanol content and modulate flavour in wines. The relationships between 172 L. thermotolerans isolates, sourced from natural and anthropic habitats worldwide, were studied using a 14-microsatellite genotyping method. The resultant clustering revealed that the evolution of L. thermotolerans has been driven by the geography and ecological niche of the isolation sources. Isolates originating from anthropic, in particular oenological environments, were genetically close, thus suggesting domestication events within the species. The phenotypic performance of the strains, assessed using a number of agar platebased growth assays with different carbon sources and physicochemical conditions, provided further support for the observed clustering. To determine whether, and to what extent, L. thermotolerans strains differ in the traits of oenological importance, and harbour signatures of domestication and/or local divergence, 94 previously genotyped strains were trialled in Chardonnay fermentations. The strains and the genetic groups were compared for their fermentation performance, production of primary and secondary metabolites and pH modulation. The common traits of L. thermotolerans strains were their glucophilic character, relatively extensive fermentation ability, low production of acetic acid and formation of lactic acid, which significantly affected the pH of the wines. An untargeted analysis of volatile compounds revealed that 58 out of 90 volatiles were affected at an L. thermotolerans strain level. Besides the remarkable extent of intra-specific diversity, results confirmed the distinct phenotypic performance of L. thermotolerans genetic groups. These observations provide further support for the occurrence of domestication events and allopatric differentiation in L. thermotolerans population.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Agriculture, Food and Wine, 2018

碩士
逢甲大學
環境工程與科學所
99
Considering the use of energy, hydrogen requires expensive storage devices, and is not suitable for car at this stage. Liquid energy sources such as ethanol and butanol are attractive. Butanol is better than ethanol in the usage with gasoline. Butanol fermentation uses pure culture and its price is subject to the production and purification costs. This study is to find out environmental and nutritional for butanol-producing bacteria. The operation conditions of seed, nutrient formulation, substrate type and concentration, and extra lactate and butyrate additions were optimized in anaerobic butanal production system using batch experiments. Moreover, a continuously stirred tank reactor (CSTR) was operated using the optimal conditions obtained from batch experiments. The results show that using sewage sludge (C.H.) seed and Che nutrient formulation (2010) with an initial pH 6.5 produced the peak butanol concentration (246 mg COD/L). An experiment was controlling initial cultivation pH to investigate sewage sludge and nutrients formulation for the optimal operating conditions. However, controlling pH continuously could enhance the butanol concentrationl to 2204 ± 1 mg COD/L which is 670% higher than the value of 286 ± 22 mg COD/L obtained from only controlling the initial cultivation pH at 6.5. Adding lactate or butyrate could not enhance the butanol fermentation in sewage sludge system. Moreover, it changed the metabolite into lactate formation. Peak butanol production concentration (3349 mg COD/L) and yields (0.039 mol butanol/mol glucose) were obtained in the operation conditions of sewage sludge (C.H.) seed, Che (2010) nutrient formula, glucose concentration 120 g/L, and control pH 6.8 h. The CSTR was operated at HRT 24 h. After 400 h operation, the reactor became stable. The performances of the reactor were food to microorganism (F/M) ratio 0.08 g glucose/g VSS-day, hydrogen content 50%, ORP -500 mV, total gas production rate 0.4 L/L/d and hydrogen production rate 0.02 L/L/d. High lactic acid concentration (99.3 g COD/L) and yield (1.55 mol lactate/mol glucose were obtained in stable period. 1, 3 - propanediol was not detected and ABE concentration was 11.7 g COD/L (acetone 0.3 g COD/L, butanol 0.1 g COD/L and ethanol 11.3 g COD/L). Key word: Butanol fermentation, Nutrient formula, pH control, Continuous system

碩士
逢甲大學
化學工程學系
103
This study conducted the investigation of 1,3-propanediol fermentation from glycerol by CSTR (continuous flow stirred tank reactor) bioreactors. First, the effect of type of nitrogen source on 1,3-propanediol (PrDOH) generation was investigated. The results showed that the PrDOH concentrations were similar when used the nitrogen source of (NH4)2SO4, NH2CONH2, or NH4HCO3, obtaining a concentration of 23.9, 23.5, and 24.4 g/L, respectively. Although there was better substrate utilization using (NH4)2SO4 as the nitrogen source, the 1,3-propanediol concentration is only 0.8 times of using NH4HCO3 as the nitrogen source. Second, the effects of HRT (hydraulic retention time, HRT) (16、12、8 and 6 h) and refined glycerol concentration (100、80、60及40 g/L) on 1,3-propanediol generation were investigated. Under the condition of refined glycerol concentration 60 g/L, the 1,3-propanediol concentration and substrate utilization decreased from 27.9 to 18.1 g/L and from 89.4 to 49.3%, respectively, with HRT decreasing from 16 to 6 h, but 1,3-propanediol production rate increased from 1.75 to 3.02 g/L/h. Under the condition of HRT 16 h, the yield of 1,3-propanediol operated at glycerol concentration 40 g/L was 0.461 g PrDOH/g glycerol consumed; it was obviously less than the values at glycerol concentration 60-100 g/L. The yield of 1,3-propanediol for the test of glycerol concentration 60, 80, and 100 g/L was 0.521, 0.550, 0.585 g PrDOH/g glycerol consumed, respectively. For these three tests, the substrate utilization decreased from 89.4 to 52.3% with glycerol concentration increasing, but the concentrations of 1,3-propanediol were similar. Under the optimum conditions (HRT 16 h and glycerol concentration 60 g/L), the concentration, yield, production rate, and utilization of 1,3-propanediol, , was 27.9 g/L, 0.521 g PrDOH/g glycerol consumed, 1.75 g/L/h, 89.4%, respectively. Third, the fermentation of 1,3-propanediol from crude glycerol was conducted at HRT 16 h and glycerol concentration 60 g/L. The results showed that the the concentration, yield and production rate was 16.1 g/L, 0.291 g PrDOH/g glycerol consumed, and 1.01 g/L/h, respectively. The performance of crude glycerol was only about 56% of refined glycerol. Finally, the effect of concentration of iron salt (FeSO4•7H2O 5-25 mg/L) on 1,3-propanediol production was investigated. The results showed that the biomass concentration in the reactor decreased with decrease of concentration of iron salt, resulting in decreasing the concentration of 1,3-propanediol. The maximum yield occurred at FeSO4•7H2O 15 mg/L and it was 0.583 g PrDOH/g glycerol consumed.

碩士
逢甲大學
化學工程學系
101
The objective of this study was acetone-butanol-ethanol (ABE) fermentation of hydrolyzate from agricultural residues by enriched mixed culture. This study developed a hydrolysis method to produce high concentration of reducing sugar from agriculture residues(bagasse, corn cob, corn stover, rice straw, or rice husk) . Two pretreatment methods (acid and enzymatic) were evaluated. The result of acid pretreatment method showed the maximum reducing sugar concentration of 131.03 g/L and furfual 13.07 g/L via over-liming treatment by Ca(OH)2 and activated carbon to remove the inhibitors. The optimal condition for enzymatic hydrolysis by T. reesei ATCC 26921 was pH 5.5 and 55℃.The yield and reducing sugar concentration obtained from solid biomass loading of 200 g/L was 54.97 % (109.93±1.50 g/L). With a high glucose/xylose ratio of 0.75 The objective of the present study was to determine the efficacy of commercial cellulase as a sole enzyme source for enzymatic of reducing sugar production from corn cob. ABE fermentation with hydrolyzate of corn cob using mixed culture MC3 can get a maximum concentration of 3.238 g/L of ABE. The MC3 mixed culture was composed of mainly Clostridium sp.as determined by PCR-DGGE analysis.

Nowadays, the replacement of petroleum products by renewable carbon is one of the great concerns in the World. Under this strategy the production of polyhydroxyalkanoates (PHA) starting from a low-cost residue shows with high potential for applications. Combining this with a mixed microbial culture (MMC) with polymer accumulation capacity allows the necessary conditions for the production of PHA at low cost. The main objective of this study was to provide a different valorisation scheme for wood residues considering a new process to be integrated into a global biorefinery approach. Pinewood bio-oil was used as the carbon substrate for the production of PHA in a three-step process: pre-fermentation, culture selection and PHA production, all systems being operated with MMC. In the acidogenic fermentation, several conditions were tested, from which the medium containing 8 g/L of sugars with a C: N: P ratio of 100:3:1 and 10 days of retention time stand out, reaching a maximum short-chain organic acid (SCOA) concentration of 6.3 g/L and an acidification degree of 0.14 gCOD/gCOD. The culture selection reactor operated under aerobic conditions was fed directly with pure bio-oil, reaching a maximum PHA content of 13.93%. Finally, after the adaptation of the culture, accumulation tests were performed in which the maximum PHA content was 35.60%, using fermented bio-oil as a substrate. It was concluded that the pinewood bio-oil is a possible substrate for PHA production.

碩士
嘉南藥理大學
保健營養系
107
Pennisetum purpureum Schumach is a modified forage species of rich phenolic substances, anthocyanins and high antioxidant properties by the Livestock Research Institute council of agriculture executive yuan. In this study, purple napiergrass extract was fermented under different culture conditions using acetic acid bacteria and yeast mixed strains of antioxidant component and. antioxidant activity. Pennisetum purpureum Schumach dried powder was extracted of hot water for 10 minutes, an after adding 10% sugar, the mixed of acetic acid bacteria and yeasts were added for 30℃ fermentation for 15 days. Experiments were carried out in different ratios (1%, 5%, 10%) and different fermentation modes (static, shake). After the fermentation, the antioxidant component were detected and analyzed (total phenolic compound content (TPC) ,total flavonoid compound content (TFC) and monomeric anthocyanins content, polymeric anthocyanins content), antioxidant capacity test (seavenging DPPH radicals ability, trolox equivalent antioxidant capacity (TEAC), ferrous reducing antioxidant capacity (FRAC) and ferrous ion chelating ability). The results showed that the pH change of purple Pennisetum extract after different ratios fermentation (1%, 5%, 10%) was reduced from 5.4~5.5 to 3.13~3.23; the antioxidant component was in the 10% group. And total flavonoid compound increased most, 5% group had the best ability to scavenge DPPH free radicals, while 1% group showed a continuous increase in antioxidant capacity ; In the shake fermentation part, the pH decreased from 5.1 to 2.42 after fermentation; the antioxidant component (TPC and TFC) increased by 208.73 mg GAE/ml, 20.23 ug RE / ml, respectively, and the polymer anthocyanin increased by 20.7% In terms of antioxidant activity, TEAC increased by 2.07 mM, DPPH free radical scavenging ability increased by 5.64%, chelating ferrous ion capacity increased by 12.33%, and reducing power was not significantly different. Therefore, this study suggests that the extract of purple Pennisetum could enhance the antioxidant substances and activities after fermentation by acetic acid bacteria and yeast mixed strains in different proportions and fermentation modes. It is hoped that the purple pennisetum extract can be developed into a functional fermented beverage to improve its applicability.

Polyhydroxyalkanoates (PHA) are fully biodegradable polyesters, synthesized by microorganisms from renewable resources, which reveal an alternative to petroleum-based plastics. Its production by mixed microbial cultures, unlike pure cultures, allows the use of industrial by-products in the absence of sterility conditions, potentially reducing process costs. The study described focused on the valorization of a waste from a brewery industry, brewer’s spent grain (BSG), through organic acids production, towards PHA production. PHA production using mixed microbial cultures involves three steps: (1) acidogenic fermentation of BSG for organic acids production, (2) selection of the mixed cultures able to accumulate PHA and (3) PHA production. In order to extract the sugars necessary to produce organic acids (phase 1), pre-treatment methods such as acid hydrolysis and subcritical water have been studied. Acid hydrolysis was selected for further experiments as the hydrolysis yield on sugars reached was higher than using subcritical water (0.152 g total sugars g BSG-1 versus 0.008 and 0.020 g total sugars g BSG-1 at 200 °C and 230 °C, respectively). Afterwards, the pH (5.0, 5.5 and 6.0) and organic loads (10, 20 and 30 gCOD L-1) effect on the acidogenic fermentation of hydrolyzed BSG was studied in batch. It was verified, regardless of the condition, that the most produced acids are acetic and butyric. As pH and organic load affects the fermentation products profile, it is possible to manipulate PHA composition. At last, the acidogenic fermentation of hydrolyzed BSG was studied in a continuous mode, using an expanded granular sludge bed reactor (EGSB). Due to the reduced operating time, it was not possible to reach stability. However, it was observed that sugars were consumed to produce organic acids. This revealed the potential of EGSB to be used for BSG acidogenic fermentation towards PHA production.

Conventional plastics play a crucial role in society and have a negative impact on the environment and, therefore, bioplastics, such as polyhydroxyalkanoates (PHAs), become sustainable alternatives due to their biocompatibility and biodegradability. In this work, the production of PHAs by mixed microbial cultures (MMC) from an industrial waste, following a circular economy logic was studied. The valorization of a waste from the coffee industry, spent coffee grounds (SCGs) was another aim of this work. SCGs are a cheap raw material, constituted by high amounts of organic compounds, such as polysaccharides and lipids, and are, therefore, considered as a good substrate for biotechnological processes. In this work, the production of PHAs followed the three-step process: first, acidogenic fermentation (AF) of SCGs occurs, then the selection of a MMC with PHAs accumulation capacity and, finally, the accumulation of biopolymer. The application of a pre-treatment facilitated the process of AF that ran in a fluidized bed biofilm reactor (FBBR). Acetic, propionic, and butyric acids were the most produced short-chain organic acids (SCOAs), and valeric acid appeared in lower concentrations. The maximum concentration of SCOAs produced was 3.25 gCOD/L, and the maximum acidification levels were 58±3.0% and 37±3.5% for the supplied sugars and the total organic carbon available, respectively. The selection step occurred in a sequential discontinuous reactor (SBR) and the operating conditions tested allowed selecting a stable PHA-accumulating MMC. Two pseudo-stationary states (PSS) were possible to define and a maximum PHAs content of 40.0 % of a copolymer of hydroxybutyrate and hydroxyvalerate was obtained, thus demonstrating the good accumulation capacity of MMC. In the last step of the work, three accumulation tests were performed in which different substrates supplied to the selected MMC were tested. The test performed with the FBBR effluent showed a maximum accumulation of 60.8 % and a volumetric yield of 0.252 gPHA/L.h. This work demonstrated the potential of SCGs as substrate to produce PHAs by MMC, opening the possibility of its implementation at the industrial level.
Os plásticos convencionais têm desempenhado um papel crucial na sociedade e têm impacto negativo no meio ambiente e, por isso, os bioplásticos, como os polihidroxialcanoatos (PHAs), tornaram-se alternativas sustentáveis devido à sua biocompatibilidade e biodegradabilidade. Neste trabalho, estudou-se a produção destes PHAs por culturas microbianas mistas (MMC), a partir de resíduos industriais, seguindo uma lógica de economia circular. A valorização de um resíduo da indústria de café, como as borras de café (SCGs), foi outro dos objetivos deste trabalho. As SCGs são uma matéria-prima barata, constituída por grandes quantidades de compostos orgânicos, como polissacáridos e lípidos, e, por isso, é considerada um bom substrato para processos biotecnológicos. Neste trabalho a produção de PHAs seguiu o processo em três passos: primeiro ocorre a fermentação acidogénica (AF) das SCGs, depois a seleção de uma cultura microbiana com capacidade de acumulação de PHA e, por fim, a acumulação de biopolímero. Verificou-se que a aplicação de um pré-tratamento facilitava o processo de AF que decorreu num reator de biofilme de leito fluidizado (FBBR). Os ácidos acético, propiónico e butírico foram os ácidos orgânicos de cadeia curta (SCOAs) mais produzidos, sendo que ácido valérico apareceu em menores concentrações. A máxima concentração de SCOAs produzidos foi de 3.25 gCOD/L e os graus de acidificação máximos foram 58 ± 3,0 % e 37 ± 3,5% em relação aos açúcares fornecidos e ao total do carbono orgânico disponível, respetivamente. O passo de seleção ocorreu num reator descontínuo sequencial (SBR) e as condições operacionais foram testadas de forma a selecionar uma cultura acumuladora de PHA estável. Foi possível definir dois estados pseudoestacionários (PSS) e obteve-se um conteúdo máximo 40,0 % de um copolímero de hidroxibutirato e hidroxivalerato, demonstrando assim a boa capacidade de acumulação da MMC. No último passo do trabalho, foram realizados três testes de acumulação em que se testaram diferentes substratos fornecidos à MMC selecionada. O teste realizado com o efluente do FBBR apresentou uma acumulação máxima de 60,8 % e uma produtividade volumétrica de 0,252 gPHA/L.h. Este trabalho demonstrou o potencial das SCGs como substrato para a produção de PHAs por MMC, abrindo a possibilidade da sua implementação a nível industrial.
Mestrado em Biotecnologia